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Image-based cell sorting: what you see is what you get
Joseph Kovac
Like separating the wheat from the Cells settled into the wells of the array, field images, the image-based method chaff, the isolation of a specific cell where they could be visualized with any allows the sorting of much more comsubset from a heterogeneous population desired microscopy technique. Kovac plex cell phenotypes than is possible can be a painstaking process. Fluoand Voldman developed software that with FACS, Voldman says. And because rescence-activated cell sorting (FACS) automatically scanned the microwell the new technique uses a weakly foallows researchers to fluorescused, low-power laser beam cently label cellular compofor cell levitation, the method nents and then collect cells is relatively gentle and does with the desired fluorescence not affect cell viability. characteristics. But because The image-based cell FACS isolates cells by measursorting method achieved ing the total fluorescence at target-cell purity of up to only one time, the method is 89% and enrichment of up to unsuitable for sorting cells on 155×. Kovac and Voldman the basis of fluorescence localare working on a next-genization or dynamics. To isolate eration microfluidic device cells with specific spatiotemA laser “fire hose” (red beam) ejects a selected cell from a miwith more complicated valve poral fluorescence charactercrowell array; the cell is then removed from the system by lateral schemes and automated istics, Joseph Kovac and Joel liquid flow. collection, which should alVoldman at the Massachusetts low even higher purity and Institute of Technology developed array and recorded fluorescence and enrichment. With the current format, an image-based cell-sorting method, bright-field images. The researchers the researchers can collect up to ~70 which is described in AC (2007, 79, manually inspected the array image and cells/h. To reduce the analysis time, the 9321–9330). marked the cells of interest, whose posi- selection of cells with desired charac“Studies that investigate protein lotions were recorded by the software. To teristics could be automated, especially calization and cellular dynamics would collect specific cells, the researchers fofor straightforward applications such as benefit the most from image-based cell cused the laser beam on the bottom of the identification of cells with nuclear sorting,” says Voldman. For example, each target cell to “levitate” the cell out fluorescence. researchers might want to isolate cells of the well with an optical scattering “The method is never going to have in which a fluorescently labeled protein force. Voldman compares the laser that the throughput of FACS, but that’s not is localized to the nucleus, cells that exlevitates the target cell to water from a what we were aiming for,” says Voldpress a fluorescent reporter at a certain “fire hose pushing up on a beach ball.” man. “We’re interested in the informatime after treatment with a chemical, The levitated cells were washed into a tion content. This technique can do or cells with a particular morphology. collection reservoir for removal with a things that flow cytometry can’t, and it FACS is unable to distinguish these pipette. will do them in a much simpler and less cell subsets. Existing microscopy-based Kovac and Voldman demonstrated expensive fashion than existing instruimaging methods—such as laser-capthe ability of their technique to purify a ments, such as LCM.” ture microdissection (LCM), optical minor population of BA/F3 pro B cells Image-based cell sorting should tweezers, and dielectrophoretic trap labeled with CellTracker Orange from prove particularly useful for studies that arrays—can accomplish spatiotemporal a 50-fold excess of CellTracker-Greenwere previously unfeasible or extremely cell sorting, but they require complex labeled cells in a >10,000-site array. In tedious, such as genetic screens based and expensive instrumentation. addition, the researchers isolated MCFon protein localization, Voldman notes. Kovac and Voldman fabricated a 7 cells with nuclear fluorescence from By studying spatiotemporal characterissimple microfluidic device composed cells with diffuse whole-cell fluorestics of heterogeneous cell populations, of a PDMS microwell array bonded cence. In this experiment, fluorescence researchers will likely gain new insights to a glass slide. The device was placed images were overlaid with bright-field into cellular dynamics. He says, “I don’t on the automated stage of a standard images to determine the positions of think that we can say we have real modupright microscope equipped with an cell nuclei. els of cells if these models don’t include inexpensive IR laser. The researchers Because cells can be sorted on the the fact that things happen over space loaded cells into the device through basis of data from multiple fluorescence and time.” a an injection valve with syringe pumps. channels in combination with bright—Laura Cassiday J a n u a r y 1 , 2 0 0 8 / A n a ly t i c a l C h e m i s t r y
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